Abstract

Objective: To analyse the infection characteristics of pathogenic bacteria in diabetic foot infection and its relationship with the degree of vascular lesions in lower extremities.

Methods: 103 cases of patients with diabetic foot complicated with infection admitted to our department from 2009 to 2014 were collected. During hospitalization, all patients underwent double lower limbs CTA or lower extremity angiography examination, and were divided into 4 groups according to the classification (A~D) of Trans-Atlantic Inter-Society Consensus (TASC). χ2 test was adopted to analyse whether there was a difference in pathogenic characteristics and drug resistance between different TASC groups. Single factor variance analysis was used to clear and definite whether there were differences between different TASC groups.

Results: There were differences in the comparison of age, duration of diabetes, ABI, TBI, percentage of neutrophils between groups, which were statistical significance (t=14.36, P=0.000; Z=30.88, P=0.001; χ2=6.76, P=0.000; χ2=11.56, P=0.006; t=56.16, P=0.039). Among the comparisons of TASC groups, with increased degree of lower extremity vascular lesions, proportion of mixed infection was gradually increased, and there were significant differences in the incidence of mixed infection (P=0.004), besides, among patients with grade D, mixed infection cases accounted for 66.7%.

Keywords

Introduction

Diabetic foot ulcer (DFU) is currently one of the main reasons
for the hospitalization treatment of diabetes patients [1]. The
occurrence of diabetic foot is closely related to nerve, vascular
diseases and infection, which can lead to changes of the
biomechanical properties in pelma of patients [2], symptoms of
which include intermittent claudication, ischemic rest pain,
ulcers and gangrene. Foot wounds are an increasingly common
problem in people with diabetes and now constitute the most
frequent diabetes-related cause of hospitalization [3]. People
with diabetes have about a 25% chance of developing a foot
ulcer in their lifetime, about half of which are clinically
infected at presentation [4]. There are many factors for poor
prognosis of diabetic foot, such as the elderly, male, heart
failure, end-stage renal disease, larger area of foot ulcer,
peripheral neuropathy, peripheral vascular diseases, infection
and so on [5]. Among them, the lower extremity vascular
disease and infection are the most important factors affecting
the diabetes foot. In diabetic foot ulcer, infection is an
important factor which influences prognosis, and even one of
the immediate causes leading to amputation and death [6]. But
the relationship between infection type of foot ulcer as well as
pathogen and vascular lesions is still not clear. Therefore, we
aimed to investigate the pathogen infection characteristics in
foot infection patients and its relationship with the degree of
lower extremity vascular lesions and provide guidance for the
healing of diabetic foot ulcers among patients with vascular
lesions in diabetic foot infection confirmed by angiography.

Objects and Methods

Research objects

103 cases of patients clearly diagnosed with diabetic foot
infection and positive secretion culture and hospitalized for
treatment in department of hypertension and endocrinology in
Daping Hospital of Third Military Medical University from
March 2009 to March 2014, of which there were 69 males and
34 females with age of (67.56 ± 11.31 y old), 8.0 (3.0, 15.0 y)
history of diabetes, and 2.0 (0.55, 10.20 months’) history of diabetic foot. Diabetic foot patients with no infection were
excluded.

Methods

Clinical data collection: The selected patients received
medical history inquiries, physical examination, completion of
admission assessment records, comprehensive evaluation and
preliminary diagnosis and treatment program conducted by
medical staff in the center during the period of hospitalization.
Cross-sectional study was applied in this study. During
hospitalization, all patients underwent double lower limbs CTA
or lower extremity angiography examination, and were divided
into 4 groups according to the classification (A~D) of Trans-
Atlantic Inter-Society Consensus (TASC) [7].

Laboratory index: In the early treatment in the center, all
patients received the monitoring of blood pressure, blood
routine, liver and kidney function, blood lipids (total
cholesterol, glycerin trilaurate, high density lipoprotein
cholesterol, low density lipoprotein cholesterol), urine routine,
24 h urine, glycosylated hemoglobin (HbA1c), foot ulcer
secretion culture and drug sensitive test and so on. Among
them, foot ulcer secretion collection: after wound cleaning and
debridement for the first time admitted to hospital, tissues with
deep infection were excised with sterile instruments, placed in
a sterilization capped container and brought to inspection in 1H. Pathogenic identification: pathogenic culture was operated
in strict accordance with the explanation of ‘National clinical
laboratory operation rules, and qualified specimens were
directly inoculated on the blood agar plate by streak separation
method. After incubation at 37°C for 24~48 h, bacterial
colonies were identified for mycobacterium by micro
biochemical dilution method. Drug sensitivity reports were
performed in strict accordance with the latest standards
recommended by the ‘American clinical laboratory
standardization committee’ (NCCLS). The susceptibility
analysis was identified by the bacterial lab and analysed as
sensitive, intermediate, and resistant.

Severity classification of diabetic foot ulcer According to
lower extremity artery angiography examination, lower
extremity vascular CTA examination (equipment in CTA
center was ‘PHILPS BrillianceiCT256 layer’) and so on,
stenosis degree and lesions length of lower extremity vascular
were cleared and defined to determine the classification. The
classification standards of Trans-Atlantic Inter-Society
Consensus (TASC) standard were adopted [8], which divided
the classification into 4 types of A-D, and patients were
divided into 4 groups according to the above.

Classification of infection degree Low-grade infection: Fester, redness and swollen, pain, hyperaesthesia, increased
skin temperature, 2 or more than 2 tubercles, or the diameter of
ethmyphitis, redness and swollen of ulcer margins<2 cm, while
infection is limited to the skin and superficial subcutaneous
tissue with no other local or systemic complications; moderate
infection: general condition of patients includes stable glucose
metabolism with 1 or more following symptoms or signs of
infection: cellulitis whose diameter is >2 cm, lymphangitis,spread of sub fascial infection, abscess, gangrene of deep
tissues (such as muscle, tendons, bones, joints, etc.) appearing;
severe infection: a systemic infection symptoms or metabolism
function disorders, such as fever, chills, confusion, etc. [9].

Statistical methods

SPSS19.0 statistical software was used. Measurement data
were expressed by c̅ ± s or interval of Median and Quartile
(MQ), single factor Analysis of Variance (ANOVA) or nonparametric
test was adopted for comparisons of multi groups,
and rate was expressed by count data, χ2 test (Chi-square test)
was used for comparisons of rate in multi groups.

Ethical consideration

The study was carried out in compliance with the Declaration
of Helsinki of the World Medical Association, and according to
a protocol approved by the Ethical Committee of Daping
Hospital, Third Military Medical University, the approval
number is 2009004. The objectives of the study were explained
to the study participants and verbal consent was obtained
before interviewing each participant.

Results

The influence of different factors on lower extremity
vascular lesions of diabetic foot patients complicated
with infection

According to the TASC classification, patients were divided
into 4 groups, which were respectively 23 cases of grade A, 16
cases of grade B, 38 cases of C grade, and 26 cases of D grade
(Table 1). There were differences in the comparison of age,
duration of diabetes, ABI, TBI, percentage of neutrophils
between groups, which were statistical significance (t=14.36,
P=0.000; Z=30.88, P=0.001; χ2=6.76, P=0.000; χ2=11.56,
P=0.006; t=56.16, P=0.039).

TASC classification

n

Male/female (case)

Age x¯ ± s, y old)

Duration of diabetes (MQ, y)

Duration of DFU (MQ, months)

Length of hospital stay (MQ, d)

Systolic pressure (x¯ ± s, mmHg)

Diastolic pressure (x¯ ± s, mmHg)

A

23

16/7

57.61 ± 12.06b

5.00 (2.00, 8.00)b

2.00 (0.23, 12.00)

12.0 (10.50, 17.50)

131.48 ± 21.49

73.13 ± 11.13

B

16

09/7

66.88 ± 10.33b

7.00 (1.00, 11.75)b

1.50 (0.40, 15.50)

13.50 (8.75, 23.25)

142.94 ± 25.26

75.31 ± 15.92

C

38

26/12

69.92 ± 9.50b

14.00 (6.25, 20.00)b

2.00 (0.50, 10.25)

12.00 (9.00, 15.00)

142.61 ± 21.35

77.68 ± 19.10

D

26

18/8

73.35 ± 7.83b

8.50 (3.25, 17.25)b

3.00 (0.85, 8.00)

13.00 (9.00, 23.00)

146.27 ± 19.37

76.15 ± 9.78

TASC classification

ABI (x¯ ± s)

TBI (x¯ ± s)

HbA1C

Hemoglobin (x¯ ± s, g/L)

Leucocyte count (MQ) ×109/L

Neutrophile granulocyte (x¯ ± s%)

A

1.04 ± 0.20b

0.78 ± 0.11b

9.52 ± 2.53

124.52 ± 23.37

7.04 (5.80, 9.71)

69.41 ± 11.99a

B

0.71 ± 0.8b

0.47 ± 0.21b

9.63 ± 1.68

119.44 ± 13.82

9.76 (6.36, 13.33)

77.99 ± 11.61a

C

0.77 ± 0.30b

0.50 ± 0.32b

9.27 ± 2.11

116.42 ± 19.47

8.65 (6.94, 12.00)

74.34 ± 11.55a

D

0.51 ± 0.38b

0.41 ± 0.34b

10.04 ± 2.76

115.98 ± 28.32

9.63 (7.66, 12.78)

77.70 ± 7.95a

TASC classification

AST (x¯ ± s, U/L)

ALT (MQ, U/L)

Scr (MQ, µmol/L)

Total cholesterol (x¯ ± s, mmol/L)

Glycerin trilaurate (x¯ ± s, mmol/L)

LDL-CL (x¯ ± s, mmol/L)

HDL-CL (x¯ ± s, mmol/L)

A

19.42 ± 8.88

17.00 (12.00, 24.70)

69.15 (61.75, 85.55)

4.30 ± 1.10

1.19 ± 0.46

2.50 ± 0.79

0.96 ± 0.31

B

15.34 ± 4.83

13.30 (10.65, 20.43)

68.35 (61.15, 82.67)

4.42 ± 1.00

1.35 ± 0.83

2.58 ± 0.77

0.97 ± 0.33

C

18.01 ± 7.79

14.50 (11.40, 19.40)

75.65 (64.95, 19.4)

4.37 ± 1.15

1.50 ± 1.12

2.83 ± 0.89

0.96 ± 0.27

D

19.77 ± 11.06

16.45 (10.06, 21.65)

75.90 (64.15, 21.65)

4.10 ± 0.98

1.19 ± 0.46

2.37 ± 0.70

0.99 ± 0.31

aP<0.05, bP<0.01, comparison among classifications

Table 1. Basic clinical data of 103 patients.

Infection characteristics of pathogenic bacteria

Based on the infection degree for classification, in diabetic foot
patients with infection collected by this center, there were 38
cases of low-grade infection (36.9%), 46 cases of moderate
infection (44.7%), and 19 cases of severe infection (18.4%). In
low-grade infection, there were 21 cases of classes C and D
patients (55.2%); in moderate infection, there were 30 cases of
classes C and D patients (65.2%); in severe infection, there
were 13 cases of classes C and D patients (68.4%). With the
aggravated degree of vascular lesions, the ratio of above
moderate infection was increasing, but there were no
significant difference between groups. According to the drug
sensitivity test, the results indicated the presence of antibiotic
resistance accounted for 61.1% in total; there were 21 cases
indicated with poor prognosis by double examination of blood
routine before discharge after the infection was not controlled
during hospitalization.

Classification according to pathogenic types

Single infection included gram-positive bacterium infection
accounting for 29.6%, gram-negative bacterium infection
accounting for 27.1%, fungal infection accounting for 4.8%,
mixed infection accounting for 38.5%. 139 strains of pathogen
were cultured, including 24 strains of Staphylococcus aureus accounted for 17.3%, 17 strains of Pseudomonas aeruginosa accounting for 12.2%; 10 strains of Enterococcus faecalis accounting for 7.2%, respectively covering the top 3. In
addition, other Staphylococcus, Streptococcus, Klebsiella
pneumoniae and Escherichia coli were also common. Multisample
χ2 test analysis showed that among the comparisons of
TASC groups, there were differences in the ratio of mixed
infection in pathogenic type, which had statistical significance
(P=0.004). With the aggravated degree of lower extremity
vascular lesions, mixed infection proportion was gradually
increased, and among patients with grade D, mixed infection
accounted for 66.7%.

Discussion

The prevalence of diabetes in China has been increased year by
year, and the compliance rate of treatment is low. The risk of
chronic complications is high, of which diabetic foot covers all
kinds of damage factors in diabetes, and due to the poor
prognosis, the long-term quality of life in patients is seriously
affected. In recent years, domestic and abroad research on
related prognostic factors for the diabetic foot patients with
infection has gradually increased. Some current research
focuses on Wagner grading of diabetic foot and infection
degree of foot ulcer as point of penetration, a number of
demographic, clinical data, laboratory index information, etc.
are selected; analysis of the above factors which affect the
prognosis of diabetic foot infection are conducted [10-12]. In
this paper, we use the TASC classification for analysis on the
relationship between lower extremity vascular lesions and
related clinical data, laboratory index, infection degree and
pathogen of selected patients, and through the analysis on foot ulcer secretion culture and results of drug sensitive test,
infection characteristics of pathogen infection is confirmed,
and its relationship with vascular lesions is analysed.

There are many factors that affect the prognosis of patients
with diabetic foot, of which two of the most important ones are
lower extremity vascular lesions and infection, and lower
extremity vascular lesions in diabetes are characterized by
lower knees vascular lesions. Diabetic foot ulcer is easily
complicated with infection, the presence of infection will
further aggravate the patient's disease condition, and two-way
interaction will lead to deterioration during the late period of
diabetic foot, amputation and even life-threatening. Domestic
study [13] showed that the pathogens of diabetic foot patients
with infection were mainly Staphylococcus aureus, Escherichia coli, Enterococcus faecalis, which had high
resistance rate to penicillin and cephalosporin. The foreign
research [14] indicated that in foot infection patients, Pseudomonas aeruginosa, Staphylococcus aureus, Colibacillus were common, and the sensitivity to penicillin and
cephalosporin showed a downward trend. This study found that
the foot ulcer patients were mainly infected by Staphylococcus
aureus, Pseudomonas aeruginosa, Enterococcus faecalis with
more resistant strains of pathogenic bacteria, which was not
consistent with the pathogenic bacteria reported in domestic
and at abroad, suggesting that the infection of foot ulcer
patients in this area was more serious, and had the
characteristics of bacteriology in this region. Among the
patients with diabetic foot infection included in this study,
patients with a variety of antibiotic resistance accounted for
61.1% of the total, indicating that the proportion of drug
resistance appearing in patients with foot infections was higher.
In the period of hospitalization, after standardized treatment,
there were 21 cases whose ulcer was not healed, and the
clinical manifestation and the result of secretion culture still
indicated that the infection had not been corrected (7 cases of
non-drug resistance, 14 cases of drug resistance), suggesting
that the prognosis of the patients with drug-resistant foot
infection was poor. Further analysis showed that the infection
was difficult to control, considering the ulcer site was often
complicated with severe circulatory disorder caused by
vascular lesion, which made the antibiotics difficult to reach
the site of infection, leading to the blood concentration of
antibiotics in the infection site decreased, and long-term use of
antibiotic made incidence rate of resistant bacteria infection
increase significantly. Foreign studies also showed that
secondary infection of drug-resistant bacteria in diabetic foot
was the main cause of final amputation[15,16]. This study also
found that there was a significant difference in mixed infection
among pathogenic types between the different groups of
TASC. Among TASCA or class B patients with lower
extremity vascular lesions, all types of bacterial infections
were common; however, in TASC, classes C and D patients,
the proportion of mixed infection increased gradually, and the
proportion of mixed infection in D patients was up to 66.7%.
With the increase of the severity degree of the vascular lesions,
the proportion of mixed infection also increased, which
suggested that the patients with high severity degree of lower vascular lesions often had multiple bacterial infections, leading
to further deterioration of diabetic foot ulcer.

Diabetic foot ulcer complicated with infection not only affects
the healing of ulcer surface, it also causes the inflammatory
reaction which is a main factor affecting systemic body. The
study found that in the comparison of each group by TASC
classification, with the aggravation of tissue circulation
ischemia degree, there were significant differences in
neutrophil percentage, and the higher the classification of
TASC, neutrophils participate in inflammation more
significantly, suggesting that patients with serious lower
extremity vascular lesions often tends to have serious infection.
Neutrophils are the dominant player of inflammation and are
involved in tissue damage, and they are at the forefront of
resisting microbial pathogens (especially purulent bacteria)
invading human tissues. When the infection of diabetic foot
occurs, they are attracted to the site of inflammation by
chemotactic substances. Because neutrophils contain a large
number of lysosomal enzymes, it can decompose bacteria and
tissue debris engulfed into the intracellular, and prevents the
spread of pathogenic microorganisms in the body. The clinical
manifestations of diabetic foot patients with infection are
diverse, such as ethmyphitis, superficial infectious ulcer of skin
(erysipelas), deep soft tissue infection (abscess, necrotizing
fasciitis), osteomyelitis, gangrene, etc. [17]. High
concentration of inflammatory factors is closely related to the
difficult wound healing of foot ulcers [18]. Therefore, it is
necessary to strengthen the anti-infective therapy and improve
the revascularization in patients with foot infection, which may
be an important means to improve the prognosis of diabetic
foot patients.

The clinical significance of this study: mixed infection in
diabetic foot has obvious correlation with the degree of lower
extremity vascular lesions in diabetic foot, and with the
aggravated degree of lower extremity vascular lesions,
proportion of mixed infection gradually increases, thus
improving vascular lesions are also part of procedures in
infection control, especially the infection which is difficult to
control may have lower extremity vascular lesions, which need
strengthened screening and treatment. In addition,
revascularization can effectively improve lower limb blood
supply of patients with arterial occlusive disease, also helps to
control infection, so as to slow down or prevent the further
deterioration of diabetic foot ulcers [19].

Limitations of this study: this study is a retrospective analysis,
which is failed to follow up long-term amputation and survival
situation of patients, and the next step should be strengthening
the follow-up observation of the above patients.